The pancreas is a compound gland comprised of endocrine, exocrine and ductal epithelial compartments that play central roles in the regulation of metabolic functions and digestive activities. Dysfunctions associated with these compartments of the pancreas lead to diabetes, pancreatitis and pancreatic cancer, respectively. Despite their diverse functions, all of the pancreatic cell-types arise from a common progenitor population derived from the foregut endoderm. In recent years, significant advances have been made in our understanding of the environmental signals that initiate the formation and differentiation of the ventral and dorsal pancreatic primordia. Furthermore, Pdx1 and Ptf1a have been identified as two key regulatory factors that cooperate to determine the fate specification of the pancreatic multipotent progenitor cells within these primordia. More recently, Foxa1 and Foxa2 have been shown to be essential for the outgrowth and differentiation of the pancreatic primoridia and the regulation of Pdx1. Beyond these factors, little is known about the molecular regulation of the initiation of the pancreatic program from the foregut endoderm. In addition, although a large number of transcription factors that are essential for pancreatic islet specification, differentiation and development have been characterized, little is known about the specification and development of the exocrine pancreas. In the studies proposed here, we will investigate the roles of two Gata factor family members, Gata4 and Gata6, in the regulation of pancreatic primordia specification and in the development and differentiation of the exocrine pancreas. Our preliminary data using conditional knockout alleles of Gata4 and Gata6 suggest they have partially redundant functions in these regulatory processes. Furthermore, we propose that the Gata proteins may cooperate with the FoxA proteins in the regulation of Pdx1, and Gata4 may cooperate with Ptf1a in the regulation of exocrine- specific gene transcription. We intend to explore the respective roles of Gata4 and Gata6 in early pancreas formation and exocrine differentiation in the following specific aims: In Specific aim 1 we will characterize Gata-mediated regulation of pancreatic primordia formation using conditional gene ablation of the Gata genes in the Sox9+ and Pdx1+ pancreatic primordia, combined with genetic lineage tracing. In Specific aim 2 we will investigate the respective roles of the Gata factors in exocrine cell development. In Specific aim 3 we will investigate the molecular role of Gata4 in pancreas- specific gene expression by exploring genetic and physical interactions between Gata4 and Ptf1a or Gata4 and Foxa2 and determining their respective cooperative activities in regulating pancreas gene expression and development. We will also use global ChIP-Seq and RNA-Seq analysis to identify the set of genes regulated by Gata4 and/or Gata6 in the pancreas. PUBLIC HEALTH RELEVANCE: The pancreas is a compound gland comprised of endocrine, exocrine and ductal epithelial compartments that play central roles in the regulation of metabolic functions and digestive activities. Dysfunctions associated with these compartments of the pancreas lead to diabetes, pancreatitis and pancreatic cancer, respectively. The studies proposed here will identify and characterize the primary regulatory pathways that contribute to the formation and function of the pancreas, especially the exocrine pancreas, to improve treatment and tissue replacement therapies of pancreatic diseases.